Project Summary Cardiac arrhythmias are a major cause of mortality in heart disease. Patients harboring loss-of-function variants in the ankyrin-B (AnkB) gene (ANK2) display severe and complex cardiac phenotypes, including sinus node dysfunction, atrial fibrillation (AF), heart rate variability (HRV), conduction defects, catecholaminergic polymorphic ventricular arrhythmia (CPVT), and/or sudden cardiac death. Moreover, previous studies have shown that common ANK2 gene variants in the general population are associated with QTc alterations and ventricular arrhythmia susceptibility, and that AnkB levels are altered in large animal models of cardiovascular disease. While these studies have provided important insight into arrhythmia mechanisms in common and acquired forms of disease, they have also identified important gaps in our understanding regarding control of heart rate and rhythm by the autonomic nervous system. Given the importance of abnormal autonomic control in cardiac arrhythmia and disease, it is essential to understand the underlying molecular pathways important for targeting of key membrane receptors/channels. This K99/R00 proposal focuses on new roles and mechanisms underlying ion channel/membrane protein regulation in human cardiac automaticity. This K99/R00 proposal covers unexpected, but directly related areas of arrhythmia biology, each directly integrating clinical, translational, and mechanistic platforms. This proposal is based on clinical and molecular data demonstrating a key and unexpected role of AnkB in regulating the assembly and targeting of the two IKACh channel subunits G-protein-activated inwardly rectifying (GIRK1 and GIRK4) that regulate cardiac `fight or flight' responses as well as atrial excitability in response to cholinergic stimuli. We identified direct AnkB/GIRK interactions and uncovered patients with arrhythmias harboring GIRK4 variants that block the interaction. As atrial arrhythmias and inappropriate heart rate are independent predictors of cardiovascular mortality and IKACh dysregulation is a major hallmark of atrial arrhythmias, these findings will have impact on both congenital and acquired forms of human atrial disease. We hypothesize that ankyrin-B plays a key unrecognized role regulating the molecular targeting and stabilization of GIRK4/GIRK1 in atria and sinoatrial node tissue, thus controlling sympathetic/parasympathetic balance to tune the heart rate. We further hypothesize that dysfunction in the ankyrin-B pathway due to reduced ankyrin-B expression or human ankyrin- B loss-of-function variants results in loss of GIRK subunit regulation and altered cardiac automaticity. We will 1) Identify molecular mechanisms for GIRK1/GIRK4 assembly and membrane targeting; 2) Identify novel roles for AnkB-based pathways in GIRK/IKACh & autonomic regulation; 3) Define roles of AnkB/GIRK4 complex in human atrial myocytes at baseline & in disease.